Table 3.

Immune and Therapeutic Effects of Focused Ultrasound Induced Hyperthermia and Thermal Stress

Disease	Model	Treatment & FUS Parameters	Immunologic Effect	Therapeutic Effect
Colon cancer	C26, (Balb/c mouse)	-Frequency: 1 MHz, Varied intensities: 1, 2, 3, 4 W/cm2, Varied pressures: 0.109, 0.154, 0.188, 0.217 MPa, MIs: 0.147, 0.207, 0.254, 0.283, Temperatures: 32-44°C. Duty cycle: 50%, Burst rate: 2 Hz, Treatment time: 2 min (thermal stress). -Bolus injection of decafluorobutane (DFB) bubble liposomes at 0.1 mg/mL, 30 L/mouse	-Mice with tumors were injected either with anti-CD8+, anti-CD4+ or anti-NK cell antibody for in-vivo depletion study. -16 days after tumor inoculation, tumors that were treated and CD4+/CD8+ depleted, CD8+ depleted or not were all significantly larger than tumors that were treated but not depleted.	-Tumoral necrosis evident at 3 and 4 W/cm2 with bubble liposome treatment -Significant tumor volume % decrease for mice treated with 3 and 4 W/cm2 and bubble liposomes. (118)
Colon cancer	C26, (Balb/c mouse)	-Hyperthermia treatment 24 hr after injection of thermosensitive liposome-laden Salmonella (groups: HIFU only, Salmonella only, Salmonella + HIFU, TB, TB+HIFU) -Pulse repetition frequency (PRF): 5 Hz, Power: 6 W, Duty cycle: 35%, Temp: 40-42.5°C, each point heated 60 s, total treatment time: 30 min.	-TB+HIFU group caused highest increase of M1 macrophages compared to other groups (without increase in M2), Salmonella and TB increased IFN- expressing CD4+/CD8+ cells with and without HIFU, no change in MDSCs in any groups. -Significantly increased cytokine levels of TNF-α (TB+HIFU) and IL-1 (TB and Salmonella) and decrease in IL-10 (TB+HIFU and Salmonella + HIFU) compared to other groups.	-Significant tumor regression with TB+HIFU group over 5 days compared to all groups. -HIFU increased apoptotic cells. (123)
Melanoma, mammary adenocarcinoma	B16F10/B16-OVA (C57BL/6 mouse), NDL (FVB/n mouse) & MMTV-PyMT (transgenic mouse)	-B16F10/B16OVA group: IV treatment of 6 mg/kg temperature-sensitive liposomes with doxorubicin and copper (CuDox-TSL) and B16-OVA heating to 42°C. -NDL group: IV treatment of CuDox-TSL, immunotherapy (IT) treatment of CpG (100 g) and tumor heating to 42°C. 3 days later, treatment of aPD-1 (200 g, i.p.). Another group repeated this treatment 4 days after. A third group used a treatment schedule of priming with CpG and aPD-1 on day 0, 3 and 7 followed by CuDox-TSL and heating to 42°C on day 10, followed by CpG and aPD-1 on day 15. -MMTV-PyMT group: Same treatment as NDL group with repeated treatment. -Pressure: 2.5 MPa, PRF: 100 Hz, burst duration: 0-7 ms controlled to maintain temperature at 42oC for 25 minutes.	-B16F10/B16OVA group: Distant B16F10 tumors experienced a significant increase in leukocytes, DCs and macrophages displaying the MHC-I-SIINFEKL complex compared to nontreated control. -NDL group: Treatment with CuDox-TSL and immunotherapy increased intratumoral CD8+ T cells and macrophages in both treated and distant tumors compared to nontreated control, however, CD8+ secretion of IFN-g did not increase. Tumors treated with immunotherapy only demonstrated significant increase of IFN-g secreting CD8+ cells. Foxp3 was not affected. Repeated dosing of CuDox-TSL and immunotherapy had decreased amount of CD8+ cells, IFN-g secreting CD8+ cells and MDSCs compared to one treatment. The third group of immunotherapy priming and single CuDox-TSL dose with 42°C ultrasound significantly enhanced CD8+ tumor infiltration. Repeated treatment of CuDox-TSL and ultrasound increased blood NK cells. - MMTV-PyMT group: repeated IT and CuDox-TSL with 42°C ultrasound dosing demonstrated extensive necrosis and enhanced CD8+ and macrophage infiltration compared to immunotherapy only.	-NDL group: 9 of 10 mice treated with IT priming, single CuDox-TSL dose and 42°C ultrasound experienced significant tumor and distant tumor regression at day 38. IT and dox mouse survival was 90% while IT only survival was 78% (101 days). (61)